title: Separation and reformation of cell surface dopamine receptor oligomers visualized in cells.
abstract: We previously showed that dopamine receptors existed as homo- and heterooligomers, in cells and in brain tissue. We developed a method designed to study the formation and regulation of G protein coupled receptor (GPCR) oligomers in cells, using a GPCR into which a nuclear localization sequence (NLS) had been inserted. Unlike wildtype GPCRs, in the presence of agonist/antagonist ligands the GPCR-NLS is retained at the cell surface, and following ligand removal, the GPCR-NLS translocated from the cell surface. The D(1) dopamine receptor expressed with either D(2)-NLS or D(1-)NLS receptors translocated to the nucleus, indicating hetero- or homo-oligomerization with the NLS-containing receptor. Using these tools, we now demonstrate that D(1)-D(2) dopamine heterooligomers can be disrupted and the component receptors separated by dopamine and selective agonists that occupied one or both binding pockets. Subsequent agonist removal allowed the reformation of the heterooligomer. D(1) receptor homooligomers could also be disrupted by agonist, but at higher concentrations than that required for the disruption of the D(1)-D(2) heteromer. Dopamine D(1) or D(2) receptor antagonists had no effect on the integrity of the homo- or heterooligomer. We have also determined that the D(1)-D(2) heterooligomer contains D(1) homooligomers. These studies indicate that the populations of dopamine receptor oligomers at the cell surface are subject to conformational changes following agonist occupancy and are likely dynamically regulated following agonist activation.
authors.affiliation: Centre for Addiction and Mental Health, University of Toronto, Toronto, Ontario, Canada. brian.odowd@utoronto.ca
authors.forename: Brian F
authors.initials: BF
authors.lastname: O'Dowd
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Ji
authors.forename: Mohammad
authors.initials: M
authors.lastname: Alijaniaram
authors.forename: Tuan
authors.initials: T
authors.lastname: Nguyen
authors.forename: Susan R
authors.initials: SR
authors.lastname: George
mesh_terms: D004298:Dopamine / Q000494:pharmacology; D018491:Dopamine Agonists / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D057054:Molecular Imaging; D019913:Nuclear Localization Signals / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects; D020836:Protein Structure, Quaternary; D017447:Receptors, Dopamine D1 / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000819:agonists / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
chemical_list: D018491:Dopamine Agonists; D019913:Nuclear Localization Signals; D017447:Receptors, Dopamine D1; D017448:Receptors, Dopamine D2; D043562:Receptors, G-Protein-Coupled; D004298:Dopamine